Sometimes, as a ribosome passes along a messenger RNA (mRNA) strand translating nucleotide triplets into proteins, it runs into a little snag, like a zipper missing a link. Secondary structures in the mRNA, which also need to be unfolded or untangled, can slow down or even stop protein synthesis. Using single-molecule FRET (fluorescence resonance energy transfer) microscopy techniques, Yale E. Goldman, MD, PhD, professor of Physiology, and Barry S. Cooperman, PhD, Professor of Chemistry, and their team recently studied this phenomenon in intricate detail impossible to obtain with other methods.
Instead of merely demonstrating that secondary structures called stem-loops or pseudoknots were somehow impeding the process, the FRET technique allowed Goldman and his team to actually determine what happens when mRNA needs to be unwound.
They found that stem-loops, composed entirely of guanine-cytosine nucleotide pairs, slowed down both transfer RNA (tRNA) motions within the ribosome and tRNA’s exit by up to 66 percent, suggesting that two steps involving the ribosome’s helicase enzyme are needed to unwind them.
But stem-loops and pseudoknots containing both GC and AU base pairs are only slowed in their dissociation from the ribosome exit site (E-site). However, these have little effect on tRNA translocation within the ribosome, which indicates that the unfolding process is more closely related to the tRNA E-site dissociation.
Many viral pathogens, for instance HIV, trick cellular ribosomes into synthesizing extra viral proteins, in part by briefly slowing translation with RNA secondary structures, giving this basic research strong health care relevance.